Keiya Nakamura

Induction of heat shock proteins and their implication in protection against ethanol-induced damage in cultured guinea pig gastric mucosal cells

The induction of heat shock proteins in cultured guinea pig gastric mucosal cells was investigated to assess their role in gastric cytoprotection. In response to sublethal heat stress at 43 degrees C for 1 hour, the cells synthesized a 72-kilodalton protein and increased the synthesis of 74- and 90-kilodalton proteins, which were detected using gel electrophoresis after [35S]methionine labeling of the cells. Immunoblot analysis indicated that the 72- and 74-kilodalton proteins were members of the heat shock protein 70 family. Northern blot analysis showed the induction of a 2.6-kilobase messenger RNA of heat shock protein 70 gene only with heat treatment. Furthermore, with heat treatment, there was significant reduction of damage after ethanol treatment. This reduction was blocked with a protein synthesis inhibitor, cycloheximide, and was associated with inhibition of synthesis of heat shock proteins. These results strongly suggest that synthesis of heat shock proteins plays an important role in the intracellular mechanism of gastric protection against ethanol.

Effect of food-restriction stress on immune response in mice

Daily 23-h food deprivation for 1-5 days induced gastric ulcers and atrophic changes of the spleen and thymus, accompanied by a rise in plasma cortisol and catecholamine levels in mice. It also modulated several immune cell functions in the spleen including a drop in the B cell population but no change in the mitogen response of the B cells, an increase in T cell population but no change in the L3T4/Lyt2 ratio and an early increase in natural killer activity and O2- production by macrophages. These effects are thought to correlate to the increase in stress-associated humoral factors and this may partly result from stress induced by food restriction.

Increased Superoxide Anion Production and Glutathione Peroxidase Activity in Peritoneal Macrophages from Autoimmune-Prone MRL/Mp-lpr/lpr Mice

We studied the release of superoxide anion (O-2) in peritoneal macrophages from autoimmuneprone MRL/Mp-lpr/lpr (MRL/l) mice. Compared to resident peritoneal macrophages from control MRL/Mp-+/+ (MRL/n) mice, macrophages from MRL/l mice exhibited an age-related increase of spontaneous and PMA-induced O-2 secretion in association with the development of the autoimmune process. Analysis of the kinetic parameters of NADPH oxidase in macrophages revealed that MRL/l macrophages were in a primed state, as shown by the decreased Km value of the oxidase for NADPH. Furthermore, we studied several key enzymes for their ability to scavenge the oxygen radicals in the macrophages. Among the enzymes studied, only glutathione peroxidase (GSH Px) activity was increased in peritoneal macrophages from MRL/l mice and this change was closely correlated with the increase in O-2 production. Thus, GSH Px activity in macrophages seems to play an important role in macrophage functions under increased oxidative stress.

The Role of Oxygen Radicals in the Pathogenesis of Gastric Mucosal Lesions Induced in Mice by Feeding-Restriction Stress

To investigate the role of oxygen radicals in the pathogenesis of gastric mucosal lesions induced by psychological stress, we exposed 3-mo-old C3H mice to feeding-restriction stress for 1 to 5 days. Serial changes in the activities of antioxidant enzymes in the gastric mucosa, together with O2- production by macrophages, were measured. The stress increased the plasma cortisol level and started to produce acute gastric lesions (AGL) on the 2nd day. Before the development of AGL, the activity of superoxide dismutase in the gastric mucosa had already decreased and the ability of O2- production by macrophages was enhanced from the 1st day. This suggests that oxygen radicals play some role in the development of AGL induced by the stress.

Ammonia Inhibits Proliferation and Cell Cycle Progression at S-Phase in Human Gastric Cells

Helicobacter pylori (Hp) has strong ureaseactivity and produces a large amount of ammonia in thestomach. In animal studies, ammonia was shown toaccelerate cell kinetics of gastric mucosa, andlong-term exposure of the stomach to ammonia leads tomucosal atrophy. To understand this process, we examinedthe effects of ammonia on the growth and cell cycleprogression of human gastric cancer cell lines (HGC-27, MKN1, MKN45) using flowcytometric analysis. Ineach cell line, ammonia inhibited the cell growth in adose-dependent manner and caused significantaccumulation of S-phase cells at a cytostatic dose. DNA synthesis of HGC-27 cells treated with ammoniawas also suppressed to about 50% of that of theuntreated cells. Similar effects were observed onaddition of ammonium chloride at the same concentration, while adjusting the pH of the media with NaOHalone to that with the cytostatic dose of ammonia didnot affect the cell cycle progression. Theseobservations indicate that ammonia induces S-phasearrest in gastric cells independently of pH.

Studies on age-related functional changes in regulatory T cells and B cells involved in the autoantibody production of MRL/MpJ − + / + mice

Age-related changes in anti-DNA autoantibody production of MRL/MpJ- +/+ mice were investigated. In lipopolysaccharide (LPS)-stimulated cultures, spleen cells of the mice showed an age-related, marked increase in the ability to produce IgG class of the autoantibody after the age of 12 months, while they showed a tendency to decrease with age in the production of IgM class of the autoantibody. Serum levels of anti-DNA autoantibodies rose markedly in the IgG autoantibody but not in the IgM autoantibody after 12 months of age, which is well consistent with the observation in the LPS-stimulated cultures. T cell-depleted spleen cells, however, showed only a small increase with age in the IgG autoantibody productive ability. These results suggest that the age-associated increase in the IgG autoantibody production in the mice is under T-cell control. Age-associated changes in suppressor capacity in spleen cells of the mice were also investigated. Suppressive activity of the cells stimulated by 2-day incubation with concanavalin A (Con A) showed a clear increase as the donor age advanced, when assayed on the LPS-stimulated anti-DNA autoantibody production in vitro. The results indicate that, in MRL/MpJ-+/+ mice, suppressor capacity does not decline with age and is not related as a cause to the autoantibody production.

Effect of aging on induction of oral tolerance by intragastric administration of sheep red blood cells in mice

Fed SRBC induced oral tolerance in young C3H mice but not in the aged. An injection of SRBC via portal vein did not tolerize C3H mice. Gut associated local immune system seems to play a key role in induction of oral tolerance and the tolerance inducing function declines with aging. Mucosal immune system plays important roles in preventing invasion by viruses, bacteria, and other parasites. Oral injection of live virus generates local and systemic immunity (1). However, fed antigen often induces oral tolerance (2). Systemic immune system in mice shows age-associated resistance to tolerance induction by tolerogen via parenteral route (3). Here, we studied oral tolerance induction by SRBC in the systemic immune system of young and aged mice.